Plant Cell Physiol. 44(7): 667–675 (2003) JSPP © 2003
Aluminum Rapidly Depolymerizes Cortical Microtubules and Depolarizes the Plasma Membrane: Evidence that these Responses are Mediated by a Glutamate Receptor Mayandi Sivaguru 1, 3, Sharon Pike 2, Walter Gassmann 2 and Tobias I. Baskin 1, 4, 5 1 2
Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, U.S.A. Department of Plant Microbiology and Pathology, University of Missouri, Columbia, MO 65211-7400, U.S.A. ;
out the world. Because aluminum is deleterious and abundant, plants have evolved mechanisms to minimize or avoid damage. Tolerance can be achieved by sequestering aluminum inside the cell in an inert form or by excreting organic anions that chelate the metal and render it harmless (Matsumoto 2000, Ma et al. 2001). While these two strategies for tolerance are probably complementary, the secretion of organic acids has received the most attention, particularly in cereals. The best understood pathway to tolerance is for maize and wheat; in these plants, exposure of roots to aluminum evokes a rapid secretion of citrate (maize) or malate (wheat). Secretion does not require de novo synthesis of the organic acid and is not limited by the activities of the relevant biosynthetic enzymes. The secretion apparently depends on plasma-membrane anion channels with appreciable conductance for organic anions, a conductance that is gated by aluminum (Ryan et al. 1997, Kollmeier et al. 2001, Piñeros and Kochian 2001, Zhang et al. 2001). In the framework of signal transduction, the secretion of organic acids is an output, a consequence of perceiving an input, namely the presence of aluminum. For aluminum, the rest of the signal transduction pathway is almost totally undefined, although parts of such a pathway are indicated by some reports. For example, in wheat, aluminum inhibits a key signal transduction enzyme, phospholipase C (Jones and Kochian 1995). Also in wheat, aluminum transiently induces a protein kinase within 30 s of exposure (Osawa and Matsumoto 2001). Finally, organic acids are secreted by rye in response to aluminum after a delay of a few h and following induction of biosynthetic enzymes, a timetable implying that a signal heralding aluminum reaches the nucleus (Li et al. 2000). To delineate the signal transduction pathway, it is necessary to characterize the events in the first min of aluminum treatment. In doing so, we followed a suggestion of Dennison and Spalding (2000) and focused on the glutamate receptor. The Arabidopsis genome contains a family of at least 20 genes homologous to animal, ionotropic glutamate receptors (Lam et al. 1998, Chiu et al. 1999, Lacombe et al. 2001). In animals, these receptors are ligand-gated cation channels, some conducting sodium and others calcium (Dingledine et al. 1999). In
Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant’s response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamatelike ligand through an anion channel and the binding of this ligand to a glutamate receptor. Keywords: Aluminum — Arabidopsis thaliana — Cortical microtubules — Membrane potential — Ionotropic glutamate receptors — Roots. Abbreviations: AP-5, 2-amino-5-phosphonopentanoate; GFP, green fluorescent protein; MBD, microtubule-binding domain; NMDA, Nmethyl-D-aspartate; NPPB, 5-nitro-2-(3¢-phenylpropyl-amino)-benzoate.
Introduction Aluminum in soil limits agricultural productivity through3 4 5
Present address: Molecular Cytology Core Facility, 2 Tucker Hall, University of Missouri, Columbia, MO 65211-7400, U.S.A. Present address: Biology Department, University of Massachusetts, Amherst, MA 01003, U.S.A. Corresponding author: E-mail,
[email protected]; Fax, +1-413-545-3243. 667
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Glutamate in the response to aluminum
Arabidopsis, at least some of these receptors may be plasmamembrane calcium channels. Symptoms of calcium deficiency were observed in a transgenic plant in which expression of one family member was obliterated (Kim et al. 2001); moreover, cytosolic calcium was elevated transiently by glutamate, apparently following calcium influx across the plasma membrane (Dennison and Spalding 2000). In view of the central relevance of calcium transients in plant signal transduction (Knight 2000), a ligand-gated calcium channel is a good candidate for a governor of signal transduction. Here, in living root cells of intact Arabidopsis seedlings, we image cortical microtubules and quantify plasma-membrane potential. We show that treatment with aluminum or glutamate depolymerizes cortical microtubules and depolarizes the membrane. Both responses are blocked by a specific antagonist of ionotropic glutamate receptors whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. As a working model, we hypothesize that endogenous glutamate effluxes through an anion channel and binds its receptor to transduce the cell’s response to aluminum.
Results Aluminum and glutamate elicit similar long-term root growth responses To study a rapid and cellular response to aluminum, we took advantage of an Arabidopsis transgenic line, GFP-MBD (green fluorescent protein–microtubule-binding domain), that allows microtubules to be imaged in living root cells (Marc et al. 1998, Granger and Cyr 2001). First, we determined that the seedling primary root in this line elongated comparably to wild type, and that elongation and root diameter were affected by aluminum comparably in the two genotypes (Fig. 1A). Aluminum requires relatively acidic pH to exert toxic effects on plants, and all media were prepared at pH 4.5, considered optimal for experiments on aluminum (Matsumoto 2000); at this pH, in the absence of aluminum, roots of both genotypes elongated vigorously (the rates for controls shown in Fig. 1 are higher than often reported for Arabidopsis at any pH). A concentration of 100 mM aluminum elicited about half maximal response, and this dose, rather than a saturating dose, was chosen for further experiments to minimize potential toxicity. At that concentration in our medium, the activity of the monomeric, trivalent species (presumably the most toxic) cannot be calculated exactly, but has been estimated to be about 10 mM by T.B. Kinraide (U.S. Department of Agriculture, Beaver WV, personal communication) who replicated the preparation of our media and performed ferron assays (Kinraide and Sweeney 2001) over the time period of our experiments. To determine whether glutamate-receptor homologs may be involved in the response to aluminum, we used exogenous glutamate to stimulate the receptor. Glutamate inhibited root elongation and stimulated radial expansion, and the response
Fig. 1 Growth responses of the primary root of Arabidopsis to (A) aluminum and (B) glutamate. Seven-day-old seedlings were transplanted onto the indicated concentration and elongation rate and root diameter measured after 24 h. Data are means ± SE of three replicate plates from a single, representative, experiment.
was similar in both genotypes (Fig. 1B). In contrast, root elongation was not affected by 5 mM aspartate (not shown). Compared with aluminum, glutamate evoked a weaker response and, at the lowest dose, glutamate inhibited elongation without affecting root diameter. Nevertheless in general the growth responses of glutamate-treated roots resembled the responses of roots treated with aluminum, with both compounds inhibiting elongation and stimulating radial expansion. A dose of 5 mM glutamate was chosen for further experiments. If aluminum and glutamate act in different pathways then both treatments given together should produce an additive effect. Instead, combining 5 mM glutamate with 100 mM aluminum inhibited elongation and increased diameter to the same extent as glutamate alone (Table 1), which is consistent with these compounds acting within a single pathway. In fact, the similarity of the responses to glutamate and to glutamate-plus-aluminum suggests that glutamate is epistatic to aluminum, implying that glutamate is downstream of aluminum, but the similarity may be coincidental. Note that in the medium used here, glutamate has been calculated to chelate monomeric, trivalent aluminum minimally (T.B. Kinraide, personal communication). Aluminum and glutamate depolymerize cortical microtubules In the GFP-MBD plants, cortical microtubules were reliably imaged throughout the root growth zone; for consistency,
Glutamate in the response to aluminum
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Table 1 Comparison of long-term growth responses to aluminum and glutamate given singly and in combination Treatment Control 100 mM aluminum 5 mM glutamate Aluminum + glutamate
Elongation rate (mm d–1) 6.9±0.3 a 3.0±0.5 b 5.0±0.1 c 4.7±0.3 c
Diameter (mm) 152±0.3 a 199±2.0 b 165±1.9 c 165±1.9 c
One-week-old seedlings were transplanted onto treatment plates and the elongation rate measured over the following 24 h and root diameter after 24 h. Data are means ± S.E. for three replicate plates with about 10 seedlings per plate. For each column, different superscript letters indicate equivalence of means rejected at P
